Compositions exhibiting ADP-ribosyltransferase activity and methods for the preparation and use thereof

ABSTRACT

Compositions characterized by ADP-ribosyltransferase activity are useful in promoting prophylactic and/or therapeutic responses as are promoted by, e.g., pertussis toxin but directed against another target antigen (e.g., a cancer-related antigen) in a mammalian patient.

CROSS-REFERENCE WITH RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 09/510,664, filed Feb. 22, 2000, now U.S. Pat. No. 6,514,499which is a divisional of U.S. patent application Ser. No. 08/482,758,now U.S. Pat. No. 6,056,960, filed Jun. 7, 1995.

BACKGROUND OF THE INVENTION

The present invention relates generally to the fields of medicine andbiology. In particular, the present invention is directed towardscompositions exhibiting ADP-ribosyltransferase activity which haveprophylactic and/or therapeutic activity (e.g., in preventing cancermetastasis, preventing recurrence or reducing the incidence of cancers),as well as methods for the preparation and use thereof.

The use of surgery and radio- or chemotherapy to treat cancer involvesthe risk of serious side-effects and even death, yet frequently fails toproduce substantive benefit. It is not surprising that these methods arerarely used to prevent cancer. It is clear there is a need for bettermethods to prevent or cure cancer and/or ameliorate the symptoms thereofin a patient.

Immune responses can effectively kill cells that display antigens thatmark cells as harboring a pathogen. Vaccines containing such antigenscan stimulate these desired responses and protect against disease withlittle risk. Coupling this experience with the hypothesis that malignantcells may also present a similar marker has led many investigators tosearch for vaccines that could prevent or cure various types of cancer[McCall, C. A., Wiemer, L., Baldwin, S., & Pearson, F. C. (1989)Bio/technology 7, 231-240; Rosenburg, S. A. (1992) J. Clin. Oncol. 10,180-199; Prehn, R. T. (1993) Proc. Natl. Acad. Sci. U.S.A. 90,4332-4333]. The successful development of such a vaccine would involveidentifying preparations containing tumor-associated antigens, andlearning how to prompt the immune system to properly and specificallykill cells displaying those antigens.

Some vaccines have been spectacularly successful at preventinginfectious disease (e.g., smallpox); attempts to make other vaccineshave, to date, failed (e.g., AIDS). At times, the lack of success mayarise from a failure to elicit a proper response to an antigen, not theunavailability of a suitable antigen. These failures suggest thatmethods that control immune responses to antigens could greatly benefitthe performance of vaccines designed to prevent, treat and/or cureinfectious disease.

Similar issues face the development of cancer vaccines. For example,injecting irradiated tumor cells frequently fails to elicit an effectiveanti-tumor response. However, injecting irradiated tumor cellspreviously transfected with genes causing production of lymphokines(e.g., GM-CSF) [Dranoff, G., Jaffee, E., Lazenby, A., Golumbeck, P.,Levitsky, H., Brose, K., Jackson, V., Hamada, H., Pardoll, D., &Mulligan, R. C. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3539-3543] canpromote anti-tumor responses. Similar results have been obtained withtumor cells transfected to produce foreign major histocompatibilitycomplexes [Plautz, G. E., Yang, Z. Y., Wu, B. Y., Gao, X., Huang, L., &Nabel, G. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4645-4649], oradhesins, such as B7, normally found on the surface ofantigen-presenting cells [Chen, L., Ashe, S., Brady, W. A., Hellstroem,I., Hellstroem, K. E., Ledbetter, J. A., McGowan, P., & Linsley, P. S.(1992) Cell 71, 1093-1102; Schwarz, R. H. (1992) Cell 71, 1068-1068;Baskar, S., Ostrand-Rosenburg, S., Nabavi, N., Nadler, L. M., Freeman,G. J., & Glimcher, L. H. (1993) Proc. Natl. Acad. Sci. U.S.A. 90,5687-5690; Townsend, S. E., & Allison, J. P. (1993) Science. 259,368-370]. Yet another approach has been to stimulate the immune systemwith bacteria or factors derived therefrom [McCall et al., 1989, supra].

Pertussis toxin is a protein released from the bacterium Bordetellapertussis. The administration of pertussis toxin along with a properantigen markedly enhances antigen-specific autoimmune disease [Munoz, J.J. (1988) in Pathogenesis and Immunity in Pertussis (Wardlaw, A. C., &Parton, R., Eds.) Chapter 8, pp. 173-192, John Wiley & Sons Ltd., NewYork; Kamradt, T., Soloway, P. D., Perkins, D. L., & Gefter, M. L.(1991) J. Immunol. 147, 3296-3302] and antigen-specific delayed-typehypersensitivity reactions, but not antigen-independent inflammatoryresponses [Sewell, W. A., Munoz, J. J., & Vadas, M. A. (1983) J. Exp.Med. 157, 2087-2096; Sewell, W. A., Munoz, J. J., Scollay, R., & Vadas,M. A. (1984) J. Immunol. 133, 1716-1722]. There are reports [Likhite, V.V. (1983) U.S. Pat. No. 4,372,945; Minagawa, H., Kakamu, Y., Yoshida,H., Tomita, F., Oshima, H., & Mizuno, D. I. (1988) Jpn. J. Cancer Res.79, 384-389; Minagawa, H., Kobayashi, H., Yoshida, H., Teranishi, M.,Morikawa, A., Abe, S., Oshima, H., & Mizuno, D. I. (1990) Br. J. Cancer62, 372-375] that crude preparations of B. pertussis can causeanti-tumor responses; the factor in these preparations causing thiseffect was not identified. Others have shown that lipopolysaccharidesfrom B. pertussis can stimulate anti-tumor responses [Ohnishi, M.,Kimura, S., Yamazaki, M., Abe, S., & Yamaguchi, H. (1994) Microbiol.Immunol. 38, 733-739; Ohnishi.M, Kimura, S., Yamazaki, M., Oshima, H.,Mizuno, D.-I., Abe, S., & Yamaguchi, H. (1994) Br. J. Cancer 69,1038-1042].

It is an object of the present invention to provide compositions andmethods which do not suffer from the drawbacks attendant to theheretofore-available compositions and methods. In particular, it is anobject of the present invention to provide compositions which increasethe efficacy of other compositions and methods.

SUMMARY OF THE INVENTION

In accordance with the present invention, there are providedcompositions characterized by ADP-ribosyltransferase activity. Thesecompositions are useful in promoting prophylactic and/or therapeuticresponses as are promoted by, e.g., pertussis toxin but directed againstanother target antigen (e.g., a cancer-related antigen) in a mammalianpatient.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be better understood with reference to theaccompanying drawings, in which:

FIG. 1 illustrates the protection against B16 melanoma afforded byvaccination of C57/BL mice with irradiated B16 tumor cells with orwithout coadministration of pertussis toxin (PT lots 48A and 55A);

FIG. 2 illustrates the protection against B16 melanoma afforded byvaccination of C57/BL mice with irradiated B16 melanoma cells firstincubated either with or without interferon-gamma (IFNg) and injectedeither with or without coadministration of pertussis toxin (PT);

FIG. 3 illustrates the protection against line 1 carcinoma afforded byvaccination of BALB/C mice with irradiated line 1 cells with or withoutcoadministration of PT:

FIG. 4 illustrates the protection against line 1 carcinoma transfectedto produce ovalbumin (L1-Ova) afforded by vaccination of BALB/C micewith irradiated L1-Ova cells with or without coadministration of PT;

FIG. 5 illustrates the ear-swelling response to irradiated L1-Ova cellsof BALB/C mice previously vaccinated with irradiated carcinoma cellswith or without coadministration of PT, or spleen cells incubated eitherwith or without pertussis toxin and then mixed with the anti-pertussistoxin monoclonal antibody 3CX4;

FIG. 6 illustrates the protection against L1-Ova cells afforded by avaccination of BALB/C mice with irradiated L1-Ova cells with or withoutthe conadministration of pertussis toxin, or spleen cells incubated withor without pertussis toxin and mixed with 3CX4; and

FIG. 7 illustrates the protection against B16 melanoma afforded byvaccination of C57/BL mice with irradiated B16 cells with or without thecoadministration of pertussis toxin, or spleen cells incubated eitherwith or without pertussis toxin and then mixed with 3CX4.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Pursuant to the present invention, responses as are promoted bypertussis toxin are directed against one or more target antigens byadministration to a mammalian patient of an effective amount of acomposition in accordance with the present invention characterized byADP-ribosyltransferase activity. Pertussis toxin is shown herein toincrease the efficacy of cancer vaccines. Compositions in accordancewith the present invention characterized by ADP-ribosyltransferaseactivity also potentiate the activity of other vaccines and other typesof therapeutic agents.

Pertussis toxin is a multi-subunit protein comprised of an A protomerconsisting of a single catalytic S1 subunit, and a B oligomer containingone S2, one S3, two S4, and one S5 subunits. The B oligomer binds thetoxin to specific receptors on target cells, thus delivering the S1subunit to the cell membrane where, after it is activated, it catalyzesthe transfer of ADP-ribose from NAD to a specific cysteine residue inspecific acceptor proteins, typically the alpha subunit of regulatoryproteins, termed G-proteins, that bind guanine nucleotides [Ui, M.(1990) in ADP-Ribosylating Toxins and G Proteins (Moss, J., & Vaughan,M., Eds.) Chapter 4, pp. 45-77, American Society for Microbiology,Washington, D.C.].

Although there are many other bacterial toxins known to catalyzeADP-ribosylations, to my knowledge there are no reports of otherbacterial toxins catalyzing the specific ADP-ribosylation reactionscatalyzed by pertussis toxin [Moss, J., & Vaughan, M., Eds. (1990)ADP-ribosylating Toxins and G Proteins, American Society forMicrobiology, Washington, D.C.]. Although a eukaryoticADP-ribosyltransferase activity has been described that adds ADP-riboseto cysteine residues, and perhaps to the same protein or residue as doespertussis toxin, its functional significance appears to be unknown[Williamson, K. C., & Moss, J. (1990) in ADP-Ribosylating Toxins and GProteins (Moss, J., & Vaughan, M., Eds.) pp. 493-510, American Societyfor Microbiology, Washington, D.C.]. If such an enzyme could bemanipulated to catalyze the same reaction as does pertussis toxin, thenit could become a functional analog of the toxin.

Other defined agents currently used to boost anti-tumor responses do notappear to contain the ADP-ribosyltransferase activity of pertussistoxin. In experiments involving stimulation of delayed-typehypersensitivity (DTH) or auto-immune responses, pertussis toxin iscommonly used in addition to other adjuvants, such as complete Freund'sadjuvant or superantigens [Munoz, J. J., & Sewell, W. A. (1984) Infect.Immun. 44, 637-641; Sewell, W. A., de Moerloose, P. A.,McKimm-Breschkin, J. L., & Vadas, M. A. (1986) Cell. Immunol. 97,238-247; Kamradt et al., 1991, supra]; thus, the mechanism underlyingpertussis toxin action and these other adjuvants is different.

It is presently preferred to administer to mice an amount of theexemplary active agent sufficient to cause lymphocytosis, which servesas a positive control to demonstrate that the agent is active in vivo.Lymphocytosis may not be required for the beneficial effects, andamounts less than this amount may be sufficient for other responses[Munoz, J. J., Arai, H., Bergman, R. K., & Sadowski, P. L. (1981)Infect. Immun. 33, 820-826], including the anti-tumor response,particularly if the agent is directed towards specific cellular targets.In humans, another measure of pertussis toxin action may be moreappropriate, for example enhanced insulin secretion or glucoseclearance. A single intravenous injection of 0.5 or 1.0 ug pertussistoxin protein per kg body weight has been found to promote insulinsecretion in healthy, control humans with no clearly-evident toxic oradverse response. [Toyota, T., Kai, Y., Kakizaki, M., Sakai, A., Goto,Y., Yajima, M., & Ui, M. (1980) Tohoku J. Exp. Med. 130, 105-116]. Theagent may be administered by a variety of appropriate routes (e.g.intravenously, intraperitoneally) as long as the agent reaches cellswhich, upon intoxication, provide an anti-tumor response.

In the examples given herein, the effective dose of pertussis toxininjected intraperitoneally into mice appears to be less than 400 ng permouse (typical weight of 25 gm) As would readily be appreciated by thoseskilled in the field, an optimum dose of active agent for any givenmammalian patient may be determined empirically.

Properly administered, the effective dose for purposes of the presentinvention could be no more than or even less than the amount of activitycontained in whole-cell pertussis vaccines. Such vaccines have beenshown to reduce hyperglycemia in diabetics requiring high dosage ofinsulin [Dhar, H. L., Dhirwani, M. K., & Sheth, U. K. (1975) Brit. J.Clin. Pract. 29, 119-120], perhaps by increasing secretion of insulin.Although it has been claimed that an untoward event can arise from about300,000 doses of this vaccine, many doubt that these rare events arecausally related to the vaccine, or the pertussis toxin it contains.Millions of infants are still routinely immunized with such vaccines inthe United States [Cherry, J. D., Brunell, P. A., Golden, G. S., &Karzon, D. T. (1988) Pediatrics. 81(6 Part 2), 939-984]. Thus the risksfrom suitable agents should be well accepted by, e.g., cancer victimsfacing extended periods of suffering and death.

Just as intoxication of some cells by pertussis toxin can promoteanti-tumor responses, intoxication of other cells could diminish thedesired response, or cause unwanted side-effects. Thus, identifying thetarget(s) of pertussis toxin sufficient to promote desired anti-tumorresponses leads to preferred compositions and methods contemplated aswithin the scope of the present invention which more fully exploit theactivity of the compositions of the present invention. Much is knownconcerning the structure and function of pertussis toxin; this knowledgehas arisen, in large part, from efforts to better use pertussis toxin inpertussis vaccines [Sato, H., & Sato, Y. (1984) Infect. Immun. 46,415-421; Pizza, M., Covacci, A., Bartoloni, A., Perugini, M., Nencioni,L., De-Magistris, M. T., Villa, L., Nucci, D., Manetti, R., Bugnoli, M.,Giovannoni, F., Olivieri, R., Barbieri, J. T., Sato, H., & Rappuoli, R.(1989) Science. 246, 497-500; Loosmore, S. M., Zealey, G. R., Boux, H.A., Cockle, S. A., Radika, K., Fahim, R. E. F., Zobrist, G. J., Yacoob,R. K., Chong, P. C.-S., Yao, F.-L., & Klein, M. H. (1990) Infect. Immun.58, 3653-3662; Nencioni, L., Pizza, M., Bugnoli, M., De-Magistris, T.,Di-Tommaso, A., Giovannoni, F., Manetti, R., Marsili, I., Matteucci, G.,Nucci, D., Olivieri, R., Pileri, P., Presentini, R., Villa, L.,Kreeftenberg, J. G., Silvestri, S., Tagliabue, A., & Rappuoli, R. (1990)Infect. Immun. 58, 1308-1315; Burnette, W. N. (1991) in VaccineResearch: A Series of Advances, Vol. 1 (Koff, W., & Six, H. R., Eds.)Chapter 6, pp. 143-193, Marcel Dekker, Inc., New York] and to studytransmembrane signaling [Ui, 1990, supra]. Knowledge of site(s) ofaction of pertussis toxin which enhance anti-tumor effects leads tostrategies to identify therapeutic targets of pertussis toxin andimprove its efficacy.

For example, cells involved in the immune response (e.g.antigen-presenting or antigen-recognizing cells) are plausible targets.Such cells can be incubated and intoxicated with pertussis toxin exvivo, the remaining toxin neutralized with monoclonal antibodies, andthe intoxicated cells placed back in vivo.

Mutations in the B oligomer could be used to target pertussis toxin tosufficient targets. For example, the B oligomer contains multiplebinding sites with differing specificities. Two of these sites have beenidentified, one in subunit S2, the other in subunit S3. The site in S2appears to cause the toxin to bind to lung cilia; the site in S3 appearsto cause binding to macrophages. Macrophages can contribute to immuneresponses. In addition, mutations in S2 and S3 can convert the bindingproperties of one to the other [Saukkonen, K., Burnette, W. N., Mar, V.L., Masure, H. R., & Tuomanen, E. I. (1992) Proc. Natl. Acad. Sci.U.S.A. 89, 118-122]. Such mutations could limit the binding of pertussistoxin to one or the other cell type. Such mutations could be used todirect pertussis toxin away from cells which, when intoxicated, producean undesired response, and towards cells that, when intoxicated, producea desired response. Other alterations of pertussis toxin structure whichalter its binding properties may provide analogs with greater efficacyand/or diminished undesired side-effects. Alternatively, antibodies orcytokines (e.g. interleukin-2) could be adsorbed, coupled covalently, orexpressed as fusion-proteins with pertussis toxin or analogs containingits ADP-ribosyltransferase activity to deliver this activity withgreater specificity than does the naturally-occurring B oligomer.

DNA encoding for the activity of pertussis toxin could be delivered tospecific cell types. The portions of the toxin sequence required for itsADP-ribosyltransferase and other functions is being revealed by computer[Domenighini, M., Montecucco, C., Ripka, W. C., & Rappuoli, R. (1991)Molec. Microbiol. 5, 23-31], enzymatic [Krueger & Barbieri, 1994], andx-ray crystal studies of its structure [Stein, P. E., Boodhoo, A.,Armstrong, G. D., Cockle, S. A., Klein, M. H., & Read, R. J. (1994a)Structure 2, 45-57; Stein, P. E., Boodhoo, A., Armstrong, G. D., Heerze,L. D., Cockle, S. A., Klein, M. H., & Read, R. J. (1994b) Struct. Biol.1, 591-596]. The DNA sequence of the pertussis toxin gene fromBordetella pertussis has been reported [Nicosia, A., Perugini, M.,Franzini, C., Casagli, M. C., Borri, M. G., Antoni, G., Almoni, M.,Neri, P., Ratti, G., & Rappuoli, R. (1986) Proc. Natl. Acad. Sci. U.S.A.83, 4631-4635].

Because pertussis toxin may be causing coordinated changes in theactivities of several types of cells involved in immune responses, thecompositions and methods of the present invention may profitably beemployed in combination with other approaches. For example, cyclic-AMPis thought to increase B7 expression on antigen-presenting cells[Nabavi, N., Freeman, G. J., Gault, A., Godfrey, D., Nadler, L. M., &Glimcher, L. H. (1992) Nature 360, 266-268], and expression of B7 maypromote anti-tumor responses [Chen, S.-H., Li Chen, X. H., Wang, Y.,Kosai, K.-I., Finegold, M. J., Rich, S. S., & Woo, S. L. C. (1995) Proc.Natl. Acad. Sci. U.S.A. 92, 2577-2581; Schwarz, 1992, supra; Baskar etal., 1993, supra; Townsend & Allison, 1993, supra]. Pertussis toxin canincrease cyclic-AMP, and thus may increase B7 expression. As increasingcyclic-AMP in an antigen-presenting cell likely does more than increaseexpression of B7, pertussis toxin may cause antigen-presenting cells toactivate T cells in ways that would benefit approaches based onincreasing the expression of B7.

Pertussis toxin may act at sites of antigenic stimulation by eitherpromoting the release of stimulatory lymphokines (e.g.,interferon-gamma) [Sewell et al., 1986, supra] or reducing the effectsof inhibitory factors. Therefore, the administration of pertussis toxinmight improve the effectiveness of smaller doses of lymphokines used topromote anti-tumor responses. If so, then, properly used, pertussistoxin might reduce dangerous side-effects associated with the use ofsuch lymphokines. Further, the toxin might improve the action of otheradjuvants that act by causing the release of lymphokines.

The mechanisms by which pertussis toxin promotes effectiveness of tumorvaccines might also enhance the efficacy of vaccines against cellsharboring pathogens such as parasites, bacteria, or viruses. Inaddition, the G-proteins modified by pertussis toxin mediate the actionsof a wide variety of extracellular effectors in many tissues [Furman, B.L., Sidey, F. M., & Smith, M. (1988) in Pathogenesis and Immunity inPertussis (Wardlaw, A. C., & Parton, R., Eds.) Chapter 7, pp. 147-172,John Wiley & Sons, New York; Bourne, H. R., Sanders, D. A., & McCormick,F. (1990) Nature 348, 125-132]. Thus, compositions exhibitingADP-ribosyltransferase activity may have therapeutic value in othersystems (e.g. diabetes) [Dhar et al., 1975, supra; Toyota et al., 1980,supra].

There is evidence that anti-IL4 antibodies enhance the promotion of DTHby pertussis toxin [Mu, H.-H., & Sewell, W. A. (1994) Immunology 83,639-645; Rosoff, P. M., Walker, R., & Winberry, L. (1987) J. Immunol.139, 2419-2423]. Thus, antagonists of IL4 may help promote theanti-tumor effect of compositions in accordance with the presentinvention. As more is learned about the types of immune responses thatkill tumor cells, the information will suggest other potentiallybeneficial combinations of other agents and the materials and methods ofthis invention.

The antigen may be added to the composition, or it may be found in tumorcells already in vivo. For example, tumor cells in vivo could beirradiated or treated with interferon-gamma with the simultaneousadministration of PT. Alternatively, methods introducing new genes intotumor cells in vivo may render them more immunogenic [Chen, S.-H. etal., 1995, supra; Sun, W. H., Burkholder, J. K., Sun, J., Culp, J.,Turner, J., Lu, X. G., Pugh, T. D., Ershler, W. B., & Yang, N.-S. (1995)Proc. Natl. Acad. Sci. U.S.A. 92, 2889-2893]. In all of these cases,pertussis toxin activity could then be used to promote an anti-tumorresponse against the in vivo cells.

In Example 1, a recombinant analog of pertussis toxin was used whichlacks ADP-ribosyltransferase activity, but retains a general structureequivalent to naturally-occurring pertussis toxin as evidenced by theability to agglutinate erythrocytes (indicating that the B oligomer isfunctionally intact) and to elicit antibodies that neutralizenaturally-occurring pertussis toxin [Nencioni et al., 1990, supra]. Theresult in the example demonstrates that the ADP-ribosyltransferaseactivity of pertussis toxin is required for the anti-tumor effect, butdoes not rule out a role for other activities of the toxin. For example,the B oligomer not only delivers the S1 subunit containingADP-ribosyltransferase activity to cells, but also can producebiological effects [Tamura, M., Nogimori, K., Yajima, M., Ase, K., & Ui,M. (1983) J. Biol. Chem. 258, 6756-6761; Rosoffet al., 1987, supra;Strnad, C. F., & Carchman, R. A. (1987) FEBS. Lett. 225, 16-20; Stewart,S. J., Prpic, V., Johns, J. A., Powers, F. S., Graber, S. E., Forbes, J.T., & Exton, J. H. (1989) J. Clin. Invest. 83, 234-242].

To demonstrate that the anti-tumor effects of pertussis toxin are notrestricted to one tumor type or strain of mouse, Examples 1-3, and 8demonstrate an effect against B16 melanoma in C57BL/6 mice, and Examples4, 5, and 7 demonstrate an effect against a lung carcinoma termed line1, syngeneic to Balb/c mice [Blieden, T. M., McAdam, A. J., Foresman, M.D., Cerosaletti, K. M., Frelinger, J. G., & Lord, E. M. (1991) Int. J.Cancer Supplement, vol 6, 82-89]. The results with the lung carcinomasuggests that the effect of pertussis toxin is increased if the tumor ismade more immunogenic by the expression of a foreign protein, in thiscase chicken ovalbumin.

Examples 6-8 also demonstrate that the immune system is a sufficienttarget of the toxin. The approach used was to take spleen cells from onemouse, incubate them overnight with or without pertussis toxin, and thenco-administer the spleen cells with irradiated tumor cells into asyngeneic mouse, which was then subsequently challenged with live tumorcells. A problem that can be encountered with this approach is thatpertussis toxin reversibly binds to the surface of the spleen cells.Thus, when cells incubated with pertussis toxin are injected into amouse, the toxin can be transferred from the injected cells to cells ofthe recipient mouse. This process must be blocked in order to establishthat the cultured cells are in fact a sufficient target of the toxin. Amonoclonal antibody, termed 3CX4 [Kenimer, J. G., Kim, K. J., Probst, P.G., Manclark, C. R., Burstyn, D. G., & Cowell, J. L. (1989) Hybridoma.8, 37-51], was therefore used to block transfer of the toxin to therecipient mouse.

To test the ability of the antibody to block transfer, the experimentreported in Example 6 was performed. Spleen cells were incubatedovernight either with or without pertussis toxin, washed, and mixed with3CX4. Irradiated tumor cells were then injected into mice either with orwithout co-administration of these cells. For some mice, thetoxin-treated cells were lysed by freeze-thawing prior to injection;pertussis toxin tolerates freeze-thawing [Kaslow, H. R., & Burns, D. L.(1992) FASEB J. 6, 2684-2690]. The action of the toxin and cells werethen evaluated by measuring an immune response to the tumor cells:irradiated tumor cells were injected into one ear, and the response wasdetermined by measuring the swelling of the ear. When administered withirradiated cells, the addition of toxin-treated cells increased swellingwhereas the addition of toxin-treated cells lysed by freeze thawing didnot. Similarly, in this experiment, vaccination with irradiated cellsalone was not sufficient: the addition of pertussis toxin or intacttoxin-treated cells was required. The conclusion is thus that 3CX4blocks the action of pertussis toxin bound to the membranes of spleencells.

Recently, a report [Dranoff et al., 1993, supra] demonstrated protectionof mice from B-16 melanoma using irradiated cells transfected with DNAcausing production of GM-CSF. This report also examined the protocolsused in previous studies of protection which employed tumor cellstransfected to produce other cytokines. The examination suggested thatthe lack of a critical control in these other studies created themisleading impression that these cytokines were crucial for theprotective effect. The control omitted in these previous studies wasvaccination with irradiated cells alone. Several of the examplesreported herein include this control, further confirming the utility ofthe compositions and methods of the present invention.

The invention may be better understood with reference to theaccompanying examples, which are intended for purposes of illustrationonly and should not be construed as in any sense limiting the scope ofthe present invention as defined in the claims appended hereto.

EXAMPLES

In the following examples, pertussis toxin was obtained from either ListLaboratories or the State of Michigan Department of Public Health. Therecombinant, inactive analog of pertussis toxin contained inactivatingmutations in the S1 subunit (arg7->lys and glu129->gly) [Nencioni etal., 1990, supra]. Mice were obtained from standard commercial sources.The hybridoma producing monoclonal antibody 3CX4 [Kenimer et al., 1989,supra] was a gift from Dr. James Kenimer; the antibody was purified fromascites fluid using a protein A affinity procedure (Pierce BiochemicalCo.) The amounts of pertussis toxin and antibody are stated in terms ofgrams of protein determined by colorimetric protein assay [Lowry, O. H.,Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951) J. Biol. Chem.193, 265-275; Bensadoun, A., & Weinstein, D. (1976) Anal. Biochem. 70,241-250].

The B16 melanoma cell line was studied using syngeneic C57BL/6 mice andwas obtained from Dr. Malcolm Mitchell [Staib, L., Harel, W., &Mitchell, M. S. (1993) Can. Res. 53, 1113-1121]. The line 1 carcinomaand the subline producing ovalbumin were studied using syngeneic BALB/cmice and were a gift from Dr. John Frelinger [Blieden, et al., 1991,supra]. The cells were cultured and released from dishes as described[Blieden, et al., 1991, supra; Staib et al., 1993, supra], collected bycentrifugation, and resuspended in serum-free media prior to irradiationand/or injection.

Vaccinations were performed by administering, as separateintraperitoneal injections, either vehicle or an antigen preparationconsisting of irradiated tumor cells, and/or pertussis toxin (400 ng permouse) or carrier as shown in the examples. Either before or after thesevaccinations, the mice were challenged with live tumor cells via asubcutaneous injection in the upper back.

Examples 1-3 demonstrate that pertussis toxin enhances anti-tumorresponses against B16 melanoma.

Example 1 (MLT2)

On Day 0, 100,000 B16 cells were injected subcutaneously (SQ) in theback. On Day 17, mice were given intraperitoneal (ip) injectionsconsisting of various combinations of phosphate-buffered saline (PBS),400 ng of pertussis toxin (PT) or recombinant, transferase-deficientpertussis toxin (rPT), and/or 300,000 irradiated B16 melanoma cells(ir-B16). On Days 26 and 38 a second and third set of ip injections weregiven. On Day 153, all surviving mice were challenged (SQ) with 100,000B16 cells. On Day 259, all surviving mice were again challenged (SQ)with 100,000 B16 cells. In this example, the B16 cells were culturedwith gamma-interferon for 24-48 hours prior to injection.

Mice were examined for physical evidence of tumor bulging outward fromthe back or side, and the length of the tumor was recorded to thenearest cm. Typically death occurred when tumors were greater than 2 cmin length. Death was associated with clearly-evident tumor growth. Otherdata suggested that, at times, there can be incomplete tumor take incontrol animals. Thus, to better demonstrate generation of an anti-tumorresponse, survivors were subjected to the subsequent tumor challenges onDays 153 and 259.

The data for this example are summarized in Table 1 (MLT2). Pertussistoxin stimulated an anti-tumor response.

Example 2

The data for this example are shown in FIG. 1 and show pertussis toxinstimulated an anti-tumor response. Ten days prior to tumor challenge(Day-10), six groups each containing six mice were injectedintraperitoneally (ip) with different combinations of antigen (300,000irradiated B16 cells previously treated with interferon-gamma,B16-IFNg(0.3) and 400 ng pertussis toxin (PT, from two different lotstermed 48A and 55A). On Day 0, the mice were all challenged with 100,000B16 cells not treated with gamma-interferon injected subcutaneously intothe back, just below the neck. Tumor size was scored and the date ofdeath recorded.

Example 3

The data for this example are shown in FIG. 2 and show that pertussistoxin stimulated an anti-tumor response. Ten days prior to tumorchallenge (Day-10), eight groups of six mice were injected, some with400 ng pertussis toxin (PT), and some with different antigenpreparations: irradiated B16 cells (B16) either with or without a priortreatment with interferon-gamma (IFNg), either fresh (no notation) orfrozen (frzn), and either 300,000 (0.3) or 2,000,000 (2.0) cells. On Day0, all the mice were challenged with an SQ injection in the back of100,000 B16 cells. Tumor size was scored and the date of death recorded.A second set of mice were vaccinated as shown in the figure below, butthe vaccinations were 4 days after initiation of tumor. None of thesemice were protected from the tumor.

Examples 4 and 5 demonstrate that pertussis toxin enhances anti-tumorresponses against line 1 lung carcinoma.

Example 4

The data for this example are shown in FIG. 3 and show that pertussistoxin stimulated an anti-tumor response against line 1 tumor cells.Fifteen days prior to challenging mice with an SQ injection of 50,000line 1 tumor cells, 12 mice were divided into two groups each containingsix mice. Both groups were injected (ip) with 300,000 irradiated line 1tumor cells. One group received in addition an ip injection of 400 ngpertussis toxin. The mice were observed for tumor growth and the date ofdeath recorded.

Example 5

The data for this example are shown in FIG. 4 and show that pertussistoxin stimulated an anti-tumor response. Twelve days prior tochallenging mice with an SQ injection of 50,000 line 1 tumor cellstransfected with DNA encoding ovalbumin (L1-Ova), 18 mice were dividedinto three groups each containing six mice. One group was injected (ip)with 300,000 irradiated L1-Ova tumor cells. One group received 300,000irradiated cells and an additional ip injection of 400 ng pertussistoxin. One group received neither cells nor toxin. The mice wereobserved for tumor growth and the date of death recorded.

Examples 6-8 establish that treating spleen cells with pertussis toxinis sufficient to enhance anti-tumor effects.

Example 6

The data for this example show that the monoclonal antibody termed 3CX4blocks pertussis toxin action in vivo. The assay involved measuring adelayed-type hypersensitivity (DTH) response to irradiated L1-Ova tumorcells injected into the ear of a mouse. The DTH response is seen as aswelling of the ear over a period of several days. In this example, theswelling is expressed as the difference in thickness between the earinjected with tumor cells, and the other ear which was injected with asolution containing ovalbumin.

Nine days prior to injection of irradiated L1-Ova cells into the ear,groups of six mice were injected ip with combinations of 300,000irradiated L1-Ova cells, 400 ng pertussis toxin, or spleen cells fromother, naive BALB/c mice. The spleen cells were first incubatedovernight in RPMI tissue culture media supplemented with 10% fetalbovine serum. To some of the cultured cells was added 400 ng pertussistoxin per 10⁸ spleen cells prior to the overnight incubation. The nextday, the cells were centrifuged, the culture media removed, and freshmedia added. The anti-pertussis toxin monoclonal antibody termed 3CX4[Kenimer et al., 1989, supra] was then added to some of the cells (1 mgadded per 10⁸ cells); some of the cells were also lysed by freezethawing.

Mice were then injected, ip, with 300,000 irradiated L1-Ova cells. Themice were divided into six groups of six mice each. The groups receivedadditional ip injections of either vehicle, 400 ng pertussis toxin(PT-direct), or the spleen cells incubated either with or withoutpertussis toxin and 3CX4. One set of mice received spleen cells thatwere incubated with pertussis toxin followed by 3CX4 and then were lysedby freeze-thawing (spl. cells +PT +3CX4->FT). For clarity, the datashowing the effect of freeze-thawing are extracted out of the firstpanel and shown in a second panel. The data clearly show that the effectof PT was blocked by freeze-thawing the spleen cells. Thus, the effectof pertussis toxin can be mediated by spleen cells altered by the toxinin culture, it is not required to intoxicate cells of the recipientmouse.

Example 7

The data for this example are shown in FIG. 6 and show that spleen cellsincubated with pertussis toxin stimulate an anti-tumor response againstL1-Ova tumor cells. Thirteen days prior to challenging mice with an SQinjection of 50,000 L1-Ova cells, mice were divided into groupscontaining six mice, and injected ip with either antigen (300,000irradiated L1-Ova tumor cells) and/or adjuvant. The adjuvant was either400 ng pertussis toxin, or spleen cells cultured overnight with orwithout pertussis toxin, washed, and then mixed with 3CX4 as describedin Example 6. On Day 0, the mice were challenged with tumor cells; onegroup received a dose of irradiated cells in the ear as described inExample 6. The mice were evaluated for tumor growth and the date ofdeath noted.

Example 8

The data for this example are shown in FIG. 7 and show that spleen cellsincubated with pertussis toxin stimulate an anti-tumor response againstB16 tumor cells. Fourteen days prior to challenging mice with an SQinjection of 300,000 B16 tumor cells, mice were divided into groupscontaining six mice, and injected ip with either antigen (300,000irradiated B16 tumor cells) and/or adjuvant. The adjuvant was either 400ng pertussis toxin, or spleen cells cultured overnight with or withoutpertussis toxin, washed, and then mixed with 3CX4 as described inExample 6. On Day 0, the mice were challenged with tumor cells; onegroup received a dose of irradiated cells in the ear as described inExample 6. The mice were visually inspected for evidence of tumorgrowth. Those with no evidence of tumor growth were termed “tumor-free.”In this example, some tumors regressed and then reappeared.

From the foregoing description, one skilled in the art can readilyascertain the essential characteristics of the invention and, withoutdeparting from the spirit and scope thereof, can adapt the invention tovarious usages and conditions. Changes in form and substitution ofequivalents are contemplated as circumstances may suggest or renderexpedient, and any specific terms employed herein are intended in adescriptive sense and not for purposes of limitation.

1. A method for promoting an anti-tumor response directed against atarget tumor-related antigen in a mammalian patient, said methodcomprising administering to the patient: an effective amount ofcomposition comprising at least one active agent, wherein the activeagent is selected from the group consisting of isolated pertussis toxinand analogs thereof exhibiting ADP-ribosyltransferase activity, saidagent capable of eliciting an immune response against a tumor in apatient having a tumor or previously afflicted with a tumor, a suitablecarrier or excipient, and a target tumor related antigen selected fromthe group consisting of tumor cells, irradiated cells, and tumorassociated antigens.
 2. A method according to claim 1, wherein thepatient has been previously afflicted with a tumor, and wherein theanti-tumor response is prophylactic.
 3. A method according to claim 1,wherein the anti-tumor response is therapeutic.
 4. A method forpromoting an anti-tumor response directed against a tumor-relatedantigen that is present in a mammalian patient, said method comprising:administering to the patient: an amount of a composition comprising atleast one active agent, wherein the active agent is selected from thegroup consisting of isolated pertussis toxin and analogs thereofexhibiting ADP-ribosyltransferase activity, wherein said analogcomprises the S1 subunit of pertussis toxin and effective to promote animmune response against a tumor in a patient having a rumor orpreviously afflicted with a tumor; and a suitable carrier or excipient.5. A method according to claim 4, wherein the patient has beenpreviously afflicted with a tumor, and wherein the anti-tumor responseis prophylactic.
 6. A method according to claim 4, wherein theanti-tumor response promoted is therapeutic.
 7. A method according toclaim 1, wherein the target antigen is administered prior to, during orafter administration of the composition.